Novel insecticidal proteins from ferns resemble insecticidal proteins from Bacillus thuringiensis.

Lepidopterans affect crop production worldwide. The use of transgenes encoding insecticidal proteins from Bacillus thuringiensis (Bt) in crop plants is a well-established technology that enhances protection against lepidopteran larvae. Concern about widespread field-evolved resistance to Bt proteins has highlighted an urgent need for new insecticidal proteins with different modes or sites of action. We discovered a new family of insecticidal proteins from ferns. The prototype protein from Pteris species (Order Polypodiales) and variants from two other orders of ferns, Schizaeales and Ophioglossales, were effective against important lepidopteran pests of maize and soybean in diet-based assays. Transgenic maize and soybean plants producing these proteins were more resistant to insect damage than controls. We report here the crystal structure of a variant of the prototype protein to 1.98 Å resolution. Remarkably, despite being derived from plants, the structure resembles the 3-domain Cry proteins from Bt but has only two out of three of their characteristic domains, lacking the C-terminal domain which is typically required for their activities. Two of the fern proteins were effective against strains of fall armyworm that were resistant to Bt 3-domain Cry proteins Cry1Fa or Cry2A.127. This therefore represents a novel family of insecticidal proteins that have the potential to provide future tools for pest control.

The dangerous liaisons in innate immunity involving recombinant proteins and endotoxins: Examples from the literature and the Leptospira field.

Endotoxins, also known as lipopolysaccharides (LPS), are essential components of cell walls of diderm bacteria such as Escherichia coli. LPS are microbe-associated molecular patterns that can activate pattern recognition receptors. While trying to investigate the interactions between proteins and host innate immunity, some studies using recombinant proteins expressed in E. coli reported interaction and activation of immune cells. Here, we set out to provide information on endotoxins that are highly toxic to humans and bind to numerous molecules, including recombinant proteins. We begin by outlining the history of the discovery of endotoxins, their receptors and the associated signaling pathways that confer extreme sensitivity to immune cells, acting alone or in synergy with other microbe-associated molecular patterns. We list the various places where endotoxins have been found. Additionally, we warn against the risk of data misinterpretation due to endotoxin contamination in recombinant proteins, which is difficult to estimate with the Limulus amebocyte lysate assay, and cannot be completely neutralized (e.g., treatment with polymyxin B or heating). We further illustrate our point with examples of recombinant heat-shock proteins and viral proteins from severe acute respiratory syndrome coronavirus 2, dengue and HIV, for which endotoxin contamination has eventually been shown to be responsible for the inflammatory roles previously ascribed. We also critically appraised studies on recombinant Leptospira proteins regarding their putative inflammatory roles. Finally, to avoid these issues, we propose alternatives to express recombinant proteins in nonmicrobial systems. Microbiologists wishing to undertake innate immunity studies with their favorite pathogens should be aware of these difficulties.

Occupational exposure to endotoxins and small cell lung cancer: a systematic review with meta-analysis.

The relationship of occupational exposure to endotoxins with different histologic subtypes of lung cancer has not been established. Our objective was to conduct a systematic review with meta-analysis to assess the effect of exposure to endotoxins on the development of small cell lung cancer (SCLC). A bibliographic search was conducted using MEDLINE, Embase, CENTRAL, and Web of Science databases until December 2022, including all cohort and/or case-control studies that examined occupational exposure to endotoxins and SCLC. Risk of bias was assessed using the U.S. Office of Health Assessment and Translation tool. A random effects model was applied, publication bias were assessed, and a sensitivity analysis was conducted. Four papers were selected for meta-analysis purposes. A total of 144 incident cases of SCLC and 897 population or hospital controls were included. Occupational exposure to endotoxins was considered for textile/leather industry and agricultural sector workers exposed to endotoxins originating from wool, cotton, or leather dust. Except for one study, all investigations were classified as having a low probability of risk of biases. The results of the meta-analysis were not statistically significant (pooled OR: 0.86; 95% CI:0.69-1.08). In addition, neither between-study heterogeneity (I2=0%;p=0.92) nor publication bias was observed (p=0.49). The results of the sensitivity analysis, after including five studies that assessed the risk of SCLC among textile industry and crop/livestock farm workers (not specifically exposed to endotoxins), showed a negative statistically non-significant association and low between-study heterogeneity (pooled OR: 0.90; 95% CI:0.79-1.02; I2=22%;p=0.23). Subjects exposed to occupational exposure to endotoxins seem to exhibit a negative association with the development of SCLC, although the results are not conclusive.

The Role of TRP Channels in Sepsis and Colitis.

To date, several members of the transient receptor potential (TRP) channels which provide a wide array of roles have been found in the gastrointestinal tract (GI). The goal of earlier research was to comprehend the intricate signaling cascades that contribute to TRP channel activation as well as how these receptors' activity affects other systems. Moreover, there is a large volume of published studies describing the role of TRP channels in a number of pathological disorders, including inflammatory bowel disease (IBD) and sepsis. Nevertheless, the generalizability of these results is subject to certain limitations. For instance, the study of IBD relies on various animal models and experimental methods, which are unable to precisely imitate the multifactorial chronic disease. The diverse pathophysiological mechanisms and unique susceptibility of animals may account for the inconsistency of the experimental data collected. The main purpose of this study was to conduct a comprehensive review and analysis of existing studies on transient receptor potential (TRP) channels implicating specific models of colitis and sepsis, with particular emphasis on their involvement in pathological disorders such as IBD and sepsis. Furthermore, the text endeavors to evaluate the generalizability of experimental findings, taking into consideration the limitations posed by animal models and experimental methodologies. Finally, we also provide an updated schematic of the most important and possible molecular signaling pathways associated with TRP channels in IBD and sepsis.

Endotoxin Inflammatory Action on Cells by Dysregulated-Immunological-Barrier-Linked ROS-Apoptosis Mechanisms in Gut-Liver Axis.

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut-liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.

Safety Assessment: a Comparative Analysis of Quantitative Content of Bacterial Endotoxins and Evaluation of Pyrogenicity of the Kazakhstan Vaccine QazCovid-in® against COVID-19.

We measured the levels of bacterial endotoxins in the bulk vaccine product (BVP) and finished vaccine QazCovid-in® and evaluated the effect of aluminum hydroxide (adjuvant) on the results of LAL test and pyrogenicity of samples in vivo (in rabbits receiving intravenous injection into the marginal ear vein). Administration of BVP with LPS resulted in a dose-dependent increase in body temperature in rabbits similar to that caused by LPS alone, which suggests that aluminum hydroxide in the vaccine did not affect the pyrogenic response in rabbits. Moreover, the LAL test showed that the aluminum hydroxide did not hinder LPS activity after serial dilution of samples.

Bacillus thuringiensis toxin Cyt2Aa forms filamentous oligomers when exposed to lipid membranes or detergents.

The bacterium Bacillus thuringiensis (Bt) produces insecticidal proteins during the sporulation phase. These proteins are located in parasporal crystals consisting of two delta-endotoxin classes, crystal (Cry) and cytolytic (Cyt) toxins. In vitro, Cyt toxins show cytolytic activity against bacterial and a variety of insect and mammalian cells. They bind to cell membranes with unsaturated phospholipids and sphingomyelin. Although Bt and its parasporal crystals containing both Cry and Cyt toxins have been successfully used as bioinsecticides, the molecular mechanism of action of Cyt toxins is not yet fully understood. To address this, we exposed Cyt2Aa to lipid membranes and visualized membrane disruption process using cryo-electron microscopy. We observed two types of Cyt2Aa oligomers. First, Cyt2Aa forms smaller curved oligomers on the membrane surface that become linear over time, and detach when the membrane ruptures. Similar linear filamentous oligomers were also formed by Cyt2Aa in the presence of detergents without prior exposure to lipid membranes, which exhibited attenuated cytolytic activity. Furthermore, our data suggest that Cyt2Aa adopts different conformations between its monomeric and oligomeric forms. Overall, our results provide new evidence for a detergent-like mechanism of action of Cyt2Aa rather than the pore-forming model of target membrane disruption of this important class of insecticidal proteins.

New native Bacillus thuringiensis strains induce high insecticidal action against Culex pipiens pallens larvae and adults.

Mosquitoes of many species are key disease vectors, killing millions of people each year. Bacillus thuringiensis-based insecticide formulations are largely recognized as among the most effective, ecologically safe, and long-lasting methods of managing insect pests. New B. thuringiensis strains with high mosquito control effectiveness were isolated, identified, genetically defined, and physiologically characterized. Eight B. thuringiensis strains were identified and shown to carry endotoxin-producing genes. Using a scanning electron microscope, results revealed typical crystal forms of various shapes in B. thuringiensis strains. Fourteen cry and cyt genes were found in the strains examined. Although the genome of the B. thuringiensis A4 strain had twelve cry and cyt genes, not all of them were expressed, and only a few protein profiles were observed. The larvicidal activity of the eight B. thuringiensis strains was found to be positive (LC50: 1.4-28.5 g/ml and LC95: 15.3-130.3 g/ml). Bioassays in a laboratory environment demonstrated that preparations containing B. thuringiensis spores and crystals were particularly active to mosquito larvae and adults. These new findings show that the novel preparation containing B. thuringiensis A4 spores and crystals mixture might be used to control larval and adult mosquitoes in a sustainable and ecologically friendly manner.

Quantitative analysis of culturable bacteria, levels of endotoxins, inflammatory mediators and substance P in teeth with symptomatic irreversible pulpitis and in teeth with vital normal pulp tissues.

AIM: To comparatively analyse the levels of culturable bacteria, endotoxins (LPS), tumour necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and substance P in teeth with symptomatic irreversible pulpitis (SIP) and vital normal pulp (VNP) tissues. METHODOLOGY: Thirty-two patients were included (20 teeth with SIP and 12 teeth with VNP tissues) in this cross-sectional study. Samples were collected from the full length of the root canals (microbial analysis) and periapical tissues (2 mm beyond the apex for immunological analysis), using sterile absorbent paper points. The levels of culturable bacteria (culture method), endotoxins (LAL Pyrogent 5000), TNF-α, IL-1ß and substance P (ELISA) were assessed. The Mann-Whitney test was used for comparisons between the levels of CFU/mL, LPS, TNF-α, IL-1ß and substance P in the SIP and VNP groups. The statistical analysis was performed with the significance level set at 5%. RESULTS: Culturable bacteria were recovered from all teeth with SIP. On the other hand, no positive cultures were observed in the VNP tissues group (p > .05). The levels of LPS were approximately four times higher in teeth with SIP than in teeth with VNP tissues (p < .05). Higher levels of TNF-α and substance P were detected in teeth with SIP (p < .05). On the other hand, no difference in the levels of IL-1ß was detected between the two groups (p > .05). CONCLUSION: Teeth with symptomatic irreversible pulpitis present higher levels of culturable bacteria, endotoxins, TNF-α and substance P than those with vital normal pulp tissues. On the other hand, the levels of IL-1ß were similar in teeth from both groups suggesting reduced implications of this inflammatory mediator in the early stages of infection.

Toll-like receptor-4 activation by subgingival biofilm and periodontal treatment response.

OBJECTIVES: This study aims to investigate longitudinally the activation of Toll-like receptor-4 (TLR-4) by subgingival biofilm samples before and after nonsurgical periodontal therapy (NSPT). MATERIALS AND METHODS: Forty periodontitis patients received NSPT and were reviewed 3 and 6 months post-treatment. Subgingival biofilm was sampled from 4 teeth per patient, at baseline and each follow-up time point. TLR-4 activation was determined using the HEK-BLUE™/hTLR4 system. Changes in TLR-4 activation and probing pocket depths (PPDs) were evaluated using generalised linear models, and the association between TLR-4 activation and pocket reduction (defined as 6-month PPDs ≤ 3mm) was determined using generalised estimating equations. RESULTS: At 6 months, the mean TLR-4 activation by subgingival biofilm samples was significantly reduced from 11.2AU (95%CI 7.1AU, 15.4AU) to 3.6AU (95%CI 2.3AU, 4.8AU, p < 0.001), paralleling significant reductions in mean PPDs at sampled sites. The response to NSPT was associated with longitudinal TLR-4 activation profiles, with significantly higher TLR-4 activation by subgingival biofilm obtained from sites that did not achieve pocket reduction, compared to sites at which pocket reduction was achieved. CONCLUSIONS: The activation of TLR-4 by subgingival biofilm samples was reduced after NSPT, and this reduction was significantly associated with the clinical improvements (PPD reductions) at sampled sites. CLINICAL RELEVANCE: This study demonstrated an association between the longitudinal profile of TLR-4 activation by subgingival biofilm and periodontal treatment response. Longitudinal monitoring of TLR-4 activation by subgingival biofilm may potentially identify non-responsive sites, enabling targeted additional treatment.

Barriers to the Use of Recombinant Bacterial Endotoxins Test Methods in Parenteral Drug, Vaccine and Device Safety Testing.

The Bacterial Endotoxins Test (BET) is a critical safety test that is used to detect bacterial endotoxins, which are the major contributor to fever-inducing contamination risks known as pyrogens. All parenteral therapies, including every lot of injected drugs, vaccines, medical devices, must be tested for pyrogens to ensure patient safety. Bacterial endotoxins test methods were developed as a highly sensitive detection method for bacterial endotoxins, after the discovery of a clotting cascade in horseshoe crab blood. However, horseshoe crab species are limited to some inshore coastal habitats along the Atlantic coast of the USA and others throughout Asia. Fully functional horseshoe crab clotting factors can be manufactured via recombinant protein production, and several BET methods featuring recombinant horseshoe crab proteins have now been developed for commercial use. Recombinant Bacterial Endotoxins Test (rBET) methods based on the use of recombinant Factor C (rFC) were established in the European Pharmacopoeia - however, these methods have not yet been granted compendial status in the United States Pharmacopoeia (USP). In order to facilitate dialogue between stakeholders, the Physicians Committee for Responsible Medicine hosted two virtual roundtable discussions on the perceived barriers to the use of rBET methods for US FDA requirements. Stakeholders agreed that multiple rFC-based methods have been demonstrated to have suitable analytical performance, as described in ICH Q2 on the Validation of Analytical Procedures and USP <1225> on the Validation of Compendial Procedures. United States Pharmacopoeia compendial inclusion of the rFC-based and other rBET methods was favoured, in order to reduce the additional burdens created by a lack of global harmonisation on BET testing requirements.

Chemomechanical preparation influences the microbial community and the levels of LPS, LTA and cytokines in combined endodontic-periodontal lesions: A clinical study.

BACKGROUND: This study was conducted to compare the microbiomes, the levels of lipopolysaccharides (LPS), lipoteichoic acid (LTA), and cytokines (interleukin [IL]-1ß and tumor necrosis factor-alpha [TNF-α]), before and after chemomechanical preparation (CMP) of the root canals (RC) and their associated periodontal pockets (PP) in teeth with combined EPL. MATERIALS: Samples were taken from 10 RC and PP, before and after CMP. The microbiomes (next-generation sequencing, V3-V4 hypervariable region of the 16S rRNA gene), microbiome diversity (bioinformatics analyses), LPS (limulus amebocyte lysate), LTA, IL-1ß, and TNF-α (ELISA) were evaluated. A statistical analysis was performed with significance level set at 5%. RESULTS: The most abundant phyla in both sites were Firmicutes and Proteobacteria. Comparative studies of bacterial genera species revealed that some increased and others decreased after CMP at both sites. A 3% reduction in Gram-negative bacteria (RC) and a 4% increase in Gram-positive bacteria (PP) were detected. LPS levels were 4.4 times higher in PP than in the RC. LTA was detected in all samples investigated. Higher levels of IL-1ß and TNF-α were detected in both sites at baseline. After CMP, LPS, LTA, IL-1ß and TNF-α were reduced in both sites. CONCLUSION: The microbial community in the RC and PP in teeth with combined EPL indicated a similarity between both sites. CMP effectively reduced the microbial load and the LPS levels from teeth with EPL, and consequently diminished the cytokine levels. The reduction in LTA levels in the RC and PP proved challenging.

Understanding the role of endotoxin tolerance in chronic inflammatory conditions and periodontal disease.

OBJECTIVE: This review aims to present the current understanding of endotoxin tolerance (ET) in chronic inflammatory diseases and explores the potential connection with periodontitis. SUMMARY: Subsequent exposure to lipopolysaccharides (LPS) triggers ET, a phenomenon regulated by different mechanisms and pathways, including toll-like receptors (TLRs), nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB), apoptosis of immune cells, epigenetics, and microRNAs (miRNAs). These mechanisms interconnect ET with chronic inflammatory diseases including periodontitis. While the direct correlation between ET and periodontal destruction has not been fully elucidated, emerging reports point towards the potential tolerization of human periodontal ligament cells (hPDLCs) and gingival tissues with a significant reduction of TLR levels. CONCLUSIONS: There is a potential link between ET and periodontal diseases. Future studies should explore the crucial role of ET in the pathogenesis of periodontal diseases, as evidence of a tolerized oral mucosa may represent an intrinsic mechanism capable of regulating the oral immune response. A clear understanding of this host immune regulatory mechanism might lead to effective and more predictable therapeutic strategies to treat chronic inflammatory diseases and periodontitis.

Saccharomyces cerevisiae fermentation products reduce bacterial endotoxin concentrations and inflammation during grain-based subacute ruminal acidosis in lactating dairy cows.

Subacute ruminal acidosis (SARA) is a metabolic disorder in dairy cows that is associated with dysbiosis of rumen and hindgut microbiomes, translocation of immunogenic compounds from the gut lumen into blood circulation, and systemic inflammatory response. In this study we hypothesized that Saccharomyces cerevisiae fermentation products (SCFP) attenuate the increases in ruminal and peripheral bacterial endotoxin concentrations and the inflammation resulting from repeated induction of SARA. Lactating Holstein dairy cows (parity 2 and 3+, n = 32) were fed diets with or without SCFP (all from Diamond V) and subjected to 2 episodes of SARA challenges. Cows received a basal total mixed ration (TMR) containing 34% neutral detergent fiber and 18.6% starch, dry matter (DM) basis. Treatments were randomly assigned to control (basal TMR and 140 g/d of ground corn with no SCFP) or 1 of 3 SCFP treatments: basal TMR and 14 g/d Original XPC (SCFPa), 19 g/d NutriTek (SCFPb-1×), or 38 g/d NutriTek (SCFPb-2×) mixed with 126, 121, or 102 g/d of ground corn, respectively. Treatments were implemented from 4 wk before until 12 wk after parturition. During wk 5 (SARA1) and wk 8 of lactation (SARA2), grain-based SARA challenges were conducted by gradually replacing 20% of DM of the basal TMR over 3 d with pellets containing 50% wheat and 50% barley. Ruminal fluid, fecal, and blood samples were collected weekly during Pre-SARA1 (wk 4, as baseline), Post-SARA1 (wk 7), and Post-SARA2 (wk 10 for blood and wk 12 for rumen and fecal parameters) stages, and twice a week during the challenges SARA1 and SARA2. Rumen papillae samples were taken only during Pre-SARA1 and Post-SARA2. We measured the concentrations of free lipopolysaccharides (LPS) in the rumen fluid and feces; free LPS and lipoteichoic acid (LTA) endotoxins in peripheral plasma; interleukin (IL)-1ß and IL-6 in peripheral serum; acute-phase proteins, serum amyloid A (SAA), and LPS-binding protein in peripheral plasma; haptoglobin (Hp) in peripheral serum; and myeloperoxidase (MPO) in rumen papillae. Induction of SARA episodes increased free LPS concentrations in rumen fluid and tended to increase LTA in peripheral plasma. The SARA episodes increased concentration of circulating SAA and tended to increase that of IL-1ß compared with Pre-SARA1. Induction of SARA did not affect the concentrations of circulating IL-6, Hp, and MPO. The SCFP supplementation reduced plasma concentrations of LTA and SAA and serum concentration of IL-1ß compared with control. Additionally, SCFPb-2× tended to reduce ruminal LPS in second-parity cows compared with control. Overall, SCFP supplementation appeared to stabilize the rumen environment and reduce proinflammatory status, hence attenuating adverse digestive and inflammatory responses associated with SARA episodes.

Transition metal dichalcogenide-based Janus micromotors for on-the-fly Salmonella detection.

Janus micromotors encapsulating transition metal dichalcogenides (TMDs) and modified with a rhodamine (RhO)-labeled affinity peptide (RhO-NFMESLPRLGMH) are used here for Salmonella enterica endotoxin detection. The OFF-ON strategy relies on the specific binding of the peptide with the TMDs to induce fluorescence quenching (OFF state); which is next recovered due to selectively binding to the endotoxin (ON state). The increase in the fluorescence of the micromotors can be quantified as a function of the concentration of endotoxin in the sample. The developed strategy was applied to the determination of Salmonella enterica serovar Typhimurium endotoxin with high sensitivity (limits of detection (LODs) of 2.0 µg/mL using MoS2, and 1.2 µg/mL using WS2), with quantitative recoveries (ranging from 93.7 ± 4.6 % to 94.3 ± 6.6%) in bacteria cultures in just 5 min. No fluorescence recovery is observed in the presence of endotoxins with a similar structure, illustrating the high selectivity of the protocol, even against endotoxins of Salmonella enterica serovar Enteritidis with great similarity in its structure, demonstrating the high bacterial specificity of the developed method. These results revealed the analytical potential of the reported strategy in multiplexed assays using different receptors or in the design of portable detection devices.

Comparative analysis of bacterial content, levels of lipopolysaccharides and lipoteichoic acid in symptomatic and asymptomatic endodontic infections at different stages of endodontic treatment.

OBJECTIVES: This study aimed to detect the presence of specific bacteria and to evaluate the levels of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) in symptomatic necrotic root canals associated with acute apical abscess (symptomatic group - GI). It also aimed to compare the findings with those presented by asymptomatic necrotic root canals (asymptomatic group - GII) in the different stages of the endodontic treatment. MATERIALS AND METHODS: Microbiological samples were collected from 20 root canals, including purulent collection from acute apical abscesses, before and after chemo-mechanical preparation (CMP) preparation (CMP) with chlorhexidine gel 2% and after 30 days of intracanal medication (ICM) with (Ca[OH]2) + chlorhexidine. The culture method was used to evaluate the efficacy of CMP and ICM. Nested PCR was used to detect the presence of 17 specific bacteria. Levels of LPS were measured by using limulus amebocyte lysate, whereas enzyme-linked immunosorbent assay was used for the quantification of LTA. RESULTS: CMP was effective in reducing the microbial load in both groups (P < 0.05). LPS levels were higher in GI than in GII (P < 0.05). There was a significant reduction in the LPS levels after CMP and ICM (P < 0.05) in GI and GII. LTA levels were significantly reduced in GI after ICM and in GII after CMP and ICM (both P < 0.05). Fusobacterium nucleatum and Enterococcus faecalis were frequently identified in both groups, alone or in combination with each other. CONCLUSION: Different species were detected in all stages of the endodontic treatment. CMP was able to reduce bacterial content and the levels of LPS, but not of LTA in the symptomatic group. High levels of LPS were correlated with spontaneous pain and pain to percussion in the symptomatic group. CLINICAL RELEVANCE: This clinical study showed that chemo-mechanical preparation was able to reduce bacterial load and levels of LPS, but not of LTA in the symptomatic group. Elevated levels of LPS were correlated with the presence of symptomatology.

Implications of endotoxins in wound healing: a narrative review.

Bacterial toxins are thought to play a role in delayed wound healing in critically colonised and infected wounds. Endotoxins are released from Gram-negative bacteria when they are lysed by host phagocytic cells during an immune response, or by antimicrobial agents, potentially leading to a detrimental effect on the host tissues. Endotoxins can affect all aspects of the wound healing process, leading to delayed healing and contributing to wound chronicity. Release of endotoxins by bacteria can also have serious systemic effects (for example, septic shock) that can lead to high levels of patient mortality. This review summarises the role and implications on wound healing of bacterial endotoxins, describing the impact of endotoxins on the various phases of the wound healing response. There is a paucity of in vivo/clinical evidence linking endotoxins attributed to a wound (via antibiotic treatment) or their release from infecting bacteria with parameters of delayed wound healing. Future work should investigate if this link is apparent and determine the mechanism(s) by which such detrimental effects occur, offering an opportunity to identify possible treatment pathways. This paper describes the phenomenon of antimicrobial-induced endotoxin release and summarises the use of wound dressings to reduce wound bioburden without inducing microbial death and subsequent release of endotoxins, thus limiting their detrimental effects.

Human cholesteryl ester transfer protein lacks lipopolysaccharide transfer activity, but worsens inflammation and sepsis outcomes in mice.

Bacterial lipopolysaccharides (LPSs or endotoxins) can bind most proteins of the lipid transfer/LPS-binding protein (LT/LBP) family in host organisms. The LPS-bound LT/LBP proteins then trigger either an LPS-induced proinflammatory cascade or LPS binding to lipoproteins that are involved in endotoxin inactivation and detoxification. Cholesteryl ester transfer protein (CETP) is an LT/LBP member, but its impact on LPS metabolism and sepsis outcome is unclear. Here, we performed fluorescent LPS transfer assays to assess the ability of CETP to bind and transfer LPS. The effects of intravenous (iv) infusion of purified LPS or polymicrobial infection (cecal ligation and puncture [CLP]) were compared in transgenic mice expressing human CETP and wild-type mice naturally having no CETP activity. CETP displayed no LPS transfer activity in vitro, but it tended to reduce biliary excretion of LPS in vivo. The CETP expression in mice was associated with significantly lower basal plasma lipid levels and with higher mortality rates in both models of endotoxemia and sepsis. Furthermore, CETPTg plasma modified cytokine production of macrophages in vitro. In conclusion, despite having no direct LPS binding and transfer property, human CETP worsens sepsis outcomes in mice by altering the protective effects of plasma lipoproteins against endotoxemia, inflammation, and infection.

Presepsin as a predictor of septic shock in patients with urinary tract infection.

BACKGROUND: Recently, presepsin has been reported to be a useful biomarker for early diagnosis of sepsis and evaluation of prognosis in septic patients. However, few reports have evaluated its usefulness in patients with urinary tract infections (UTI). This study aimed to evaluate whether presepsin could be a valuable marker for detecting severe sepsis, and whether it could predict the therapeutic course in patients with UTI compared with markers already used: procalcitonin (PCT) and C-reactive protein (CRP). METHODS: From April 2014 to December 2016, a total of 50 patients with urinary tract infections admitted to Gunma university hospital were enrolled in this study. Vital signs, presepsin, PCT, CRP, white blood cell (WBC) count, causative agents of urinary-tract infections, and other data were evaluated on the enrollment, third, and fifth days. The patients were divided into two groups: with (n = 11) or without (n = 39) septic shock on the enrollment day, and with (n = 7) or without (n = 43) sepsis on the fifth day, respectively. Presepsin was evaluated as a biomarker for systemic inflammatory response syndrome (SIRS) or septic shock. RESULTS: Regarding the enrollment day, there was no significant difference of presepsin between the SIRS and non-SIRS groups (p = 0.276). The median value of presepsin (pg/mL) was significantly higher in the septic shock group (p < 0.001). Multivariate logistic regression analysis showed that presepsin (≥ 500 pg/ml) was an independent risk factor for septic shock (p = 0.007). ROC curve for diagnosing septic shock indicated an area under the curve (AUC) of 0.881 for presepsin (vs. 0.690, 0.583, and 0.527 for PCT, CRP and WBC, respectively). Regarding the 5th day after admission, the median presepsin value on the enrollment day was significantly higher in the SIRS groups than in the non-SIRS groups (p = 0.006). On the other hand, PCT (≥ 2 ng/ml) on the enrollment day was an independent risk factor for SIRS. ROC curve for diagnosing sepsis on the fifth day indicated an AUC of 0.837 for PCT (vs. 0.817, 0.811, and 0.802 for presepsin, CRP, and WBC, respectively). CONCLUSIONS: This study showed that presepsin may be a good marker for diagnosing septic shock based on admission data in patients with UTI.

Evaluation of bacteriophage cocktail on septicaemia caused by colistin-resistant Acinetobacter baumannii in immunocompromised mice model.

Background & objectives: Since the bacterium, Acinetobacter baumannii (AB) has acquired resistance to almost all commercially available antibiotics, the search for alternative treatment options continues to be need of the hour. Bacteriophage therapy seems to be the most promising amongst various proposed alternatives (e.g. antimicrobial peptides, bacteriocin, probiotics, etc.). The present study, therefore, aimed to evaluate the effect of different dosages of specific phages in immunocompromised rodents in a septicaemia model caused by AB mimicking real clinical situations. Methods: The three most active and unique phages (ɸAb4, ɸAb7 and ɸAb14) were selected for this study. A constant dose (100 µl of 108 pfu/ml) of AB was given in all the experiments. Five different sets of experiments were designed: prophylactic administration of phage cocktail in the volume of 100 µl (109 pfu/ml) before and simultaneous with the bacterial challenge; and therapeutic i.e. administration of phage cocktail six, 12 and 24 h after bacterial challenge. Since there were deaths in mice when phage was given 24 h after bacterial challenge, the reduced dosage i.e. 100 µl of 107, 10[6], 105 pfu/ml of phage cocktail was also evaluated. Results: The administration of 100 µl (109 pfu/ml) of phage cocktail after six, 12 and 24 h of the bacterial challenge resulted in the mortality ranging between 20 to 60 per cent. However, no mortality could be observed with simultaneous or prophylactic administration of phages with the bacterial challenge. No mortality was observed with reduced doses of the cocktail (10[6] and10[5] pfu/ml). Interpretation & conclusions: As per the results of this study, it may be concluded that even if patients with acute infections report late to the hospital, a relatively low dose of the phage cocktail may be therapeutically beneficial.